Mutants of Streptomyces glaucescens GLA.0 which are blocked in the production of tetracenomycin C (compound 1), an anthracycline antibiotic having significant antitumor activity, accumulated several new anthracycline metabolites structurally related to compound 1 and to intermediates of its biosynthetic pathway. Through chemical and spectroscopic comparisons with the known anthracycline metabolites of the wild-type strain, we identified the two regioisomers of tetracenomycin B2 (compounds 7a and 7b), 8-demethyltetracenomycin C (compound 12), tetracenomycin D2 (compound 11), tetracenomycin E (compound 13), and the 12-naphthacenone forms of compounds 7a, 7b, and 2 (tetracenomycin D1). A hypothetical biosynthetic pathway to compound 1 is presented that is consistent with the occurrence of compounds 7b, 13, and 5 (tetracenomycin A2) and with the cosynthetic behavior of tetracenomycin C-nonproducing mutants (H. Motamedi, E. Wendt-Pienkowski, and C. R. Hutchinson, J. Bacteriol. 167:575-580, 1986).